Fluorescent light images can be generated in-vivo for imaging of physiological and molecular functions and gene expression in live biological tissues. Fluorescence imaging of small animals has been used in biological research, including research in drug discovery and in understanding disease and normal development. Fluorescence imaging has also been used to study various human tissues, for example, tissues exhibiting epithelial diseases, the human breast, joints, and human teeth.
Conventionally, fluorescent light has been used to generate images of histological slices of biological tissue using so-called fluorescence microscopy. Fluorescence microscopy is used to provide relatively high-resolution images. However, tissue sectioning used in conventional fluorescence microscopy is limited to slice thicknesses (i.e., tissue depths) on the order of half a millimeter, and therefore, conventional fluorescence microscopy is not appropriate for imaging through entire organs or through the whole body of an animal or human.
In order to provide images deeper into tissue, conventional systems and techniques have used illumination light sources that can excite near infrared (NIR) fluorescent light from fluorochromes within the tissue. The near infrared light is selected because near infrared light is only mildly absorbed by tissue (compared to visible light) and can propagate several centimeters through biological tissue. Near infrared light is used in a variety of optical imaging systems and techniques.
The most common macroscopic technique that is conventionally used for fluorescence imaging is fluorescence reflectance imaging (FRI), which is also referred to herein as fluorescence epi-illumination imaging (FEI). Epi-illumination light sources and epi-illumination imaging are further described below. In general, an epi-illumination light source generates epi-illumination light that is directed toward a surface of biological tissue. The epi-illumination light propagates upon or into the biological tissue and excites fluorescent light from fluorescent material on or within the biological tissue. To form a fluorescent epi-illumination image, the fluorescent light is collected generally on the same side of the tissue as the epi-illumination light source.
As described above, an FEI system transmits light onto and/or into biological tissue and collects the fluorescent light that is emitted back from within the tissue. In some arrangements, the emitted light is near infrared light. The emitted light can be visually inspected or it can be captured with a CCD camera or other photon detector positioned generally on the same side of the tissue as the epi-illumination light source.
A second method, which has not yet been fully utilized for research using small animals, but which has found applications in optical breast imaging, uses a transillumination light source to generate transillumination images. Similar to the above-described epi-illumination light source, a transillumination light source generates transillumination light that propagates into the tissue and excites fluorescent light from a fluorescent material on or within the biological tissue. However, unlike epi-illumination light, the transillumination light propagates entirely through the tissue. In transillumination imaging, fluorescent light is collected generally on the opposite side of the tissue from the transillumination light source.
Similar to that described above for fluorescence epi-illumination imaging, in fluorescence trans-illumination imaging, excitation light (for example, near-infrared light) from a trans-illumination light source is used to illuminate the tissue. The trans-illumination light source is used to excite fluorescent material within the tissue that, in turn, emits fluorescent light. However, in contrast to the above-described fluorescence epi-illumination arrangement, in fluorescence transillumination imaging, a CCD camera or other photon detector is positioned generally on the opposite side of the tissue from the transillumination light source. In some arrangements, the emitted light is near infrared light. Fluorescence transillumination imaging (FTI) has been used to visualize functional characteristics of cardiac muscle and in dental diagnostic practice.
In some transillumination arrangements, the transillumination light source and the light detector lie on a virtual line passing through the tissue. In some arrangements the virtual line is generally perpendicular to the tissue and, in other arrangements, the virtual line is not generally perpendicular to the tissue.
Fluorescence epi-illumination imaging (FEI) and fluorescence transillumination imaging (FTI) can result in “planar” images, which are two-dimensional images.
More advanced optical imaging systems and methods have been developed, which utilize tomographic techniques. These systems and methods operate by obtaining photonic measurements at different projections (i.e., angles or slices) to the tissue and combining the measurements using a tomographic algorithm. Tomography can provide a more accurate image than the above-described forms of planar imaging. Advantages of tomography include a superior ability for image quantification, an ability to provide two-dimensional or three-dimensional images, an ability to provide three-dimensional imaging with feature depth measurements, and higher sensitivity and higher resolution as compared to planar imaging, especially deeper in tissue. In some applications, tomography has been used in-vivo to measure enzyme up-regulation and treatment response to drugs. In these applications, tomography provides superior imaging performance compared to planar imaging. However, tomography is more complex than planar imaging, requiring more advanced instrumentation, requiring multiple illumination points (projections), which can require multiple light sources, and requiring advanced theoretical methods for modeling photon propagation in tissues.
A common assumption in conventional NIR optical tomography is that propagation in a diffuse medium has high scattering but relatively low absorption, as provided by the NIR window. This assumption has allowed derivation of a “diffusion equation” associated with a “transport equation,” by means of a “diffusion approximation,” which provides an effective tool for modeling NIR photon propagation in tissues. The transport equation is described, for example, in K. M. Case and P. F. Zweifel, “Linear Transport Theory,” Addison-Wesley, M A, (1967) and the diffusion approximation in K. Furutsu and Y. Yamada, “Diffusion Approximation for a Dissipative Random Medium and the Applications,” Phys. Rev. E 50, 3634 (1994).
While optical imaging associated with fluorochromes is described above, fluorescent proteins are also known materials that can be formed within biological tissue, and which can be excited by excitation light in order to emit fluorescent light from within a biological tissue. However, as is known, all currently available fluorescent proteins utilize excitation light having a wavelength in the visible range. Moreover, conventional fluorescent proteins emit visible fluorescent light when excited. Tomographic imaging using visible light, as provided by the conventional fluorescent proteins, is complicated by a relatively high absorption of visible light propagating in biological tissue, which results in significant attenuation.
Other, more advanced solutions (other than the above-described diffusion approximation) to the transport equation have been generated and applied to NIR optical tomography. The advanced solutions overcome inadequacies of the above-mentioned diffusion approximation. However the advanced solutions to the transport equation are generally computationally expensive and become impractical for tomographic systems having a large number of excitation light sources, resulting in large data sets.
In order to provide a plurality of images necessary for tomography, many conventional optical tomography systems use an optical switch as part of a light source assembly in order to use a single light element to project at a variety of angles or positions relative to a specimen. It is known that the optical switch generates energy losses.